pH dependent ion exchange matrix and method of use in the isolation of nucleic acids

ABSTRACT

Disclosed are pH dependent ion exchange matrices, methods of making, and methods of use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This aplication is a divisional aplication of U.S. patent applicationSer. No. 09/312,172, filed May 14. 1999, now U.S. Pat. No. 6,310,199.issued Oct. 30. 2001.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

TECHNICAL FIELD

This invention relates generally to materials and methods for isolatinga target nucleic acid, such as plasmid DNA, chromosomal DNA, total RNA,mRNA, or RNA/DNA hybrids from contaminants, such as proteins, lipids,cellular debris, and non-target nucleic acids. This invention relates,particularly, to pH dependent ion exchange matrices with the capacity toadsorb a target nucleic acid in the presence of a solution at a first pHand to desorb the target nucleic acid in the presence of a secondsolution at a second pH which is different from the first pH. Thisinvention also relates to methods of making and using such pH dependention exchange matrices in isolating target nucleic acids.

BACKGROUND OF THE INVENTION

Many molecular biological techniques such as reverse transcription,cloning, restriction analysis, amplification and sequencing require thatnucleic acids used in the techniques be substantially free ofcontaminants capable of interfering with such processing or analysisprocedures. Such contaminants generally include substances that block orinhibit chemical reactions, (e.g. substances that block or inhibitnucleic acid hybridizations, enzymatically catalyzed reactions and othertypes of reactions used in molecular biological techniques), substancesthat catalyze the degradation or depolymerization of a nucleic acid orother biological material of interest, or substances which block or maskdetection of the nucleic acid of interest. Substances of this last typecan block or mask by providing a “background” indicative of the presencein a sample of a quantity of a nucleic acid of interest, (also referredto herein as a “target nucleic acid”) when the nucleic acid of interestis not, in fact, present in the sample. Contaminants also includemacromolecular substances from the in vivo or in vitro medium from whicha target nucleic acid is isolated, macromolecular substances such asenzymes, other types of proteins, polysaccharides, or polynucleotides,as well as lower molecular weight substances, such as lipids, lowmolecular weight enzyme inhibitors, oligonucleotides, or non-targetnucleic acids. Contaminants can also be introduced into a targetbiological material from chemicals or other materials used to isolatethe material from other substances. Common contaminants of this lasttype include trace metals, dyes, and organic solvents.

Obtaining target nucleic acid sufficiently free of contaminants formolecular biological applications is complicated by the complex systemsin which the target nucleic acid is typically found. These systems,e.g., cells from tissues, cells from body fluids such as blood, lymph,milk, urine, feces, semen, or the like, cells in culture, agarose orpolyacrylamide gels, or solutions in which target nucleic acidamplification has been carried out, typically include significantquantities of contaminants from which the target nucleic acid ofinterest must be isolated before being used in a molecular biologicalprocedure.

The earliest techniques developed for use in isolating target nucleicacids from such complex systems typically involve multiple organicextraction and precipitation steps. Hazardous chemicals, such aschloroform and phenol or mixtures thereof, were used in most suchprocedures. Closed circular nucleic acid molecules, such as plasmid DNA,was typically isolated further by ultra-centrifugation of plasmid DNA inthe presence of cesium chloride and ethidium bromide. See, e.g.,Molecular Cloning, ed. by Sambrook et al. (1989), pp. 1.42-1.50.Ethidium bromide is a neurotoxin. Removal of both ethidium bromide andcesium chloride from the resulting band of plasmid DNA obtained byultracentrifugation was required before the DNA could be used indownstream processing techniques, such as sequencing, transfection,restriction analysis, or the polymerase chain reaction.

In recent years, many different matrices have been developed for use inthe isolation of nucleic acids from complex biological materials. Forexample, matrices have been developed for the isolation of nucleic acidsby ion-exchange chromatography (e.g., J. of Chromatog. 508:61-73 (1990);Nucl. Acids Research 21(12):2913-2915 (1993); U.S. Pat. Nos. 5,856,192;5,82,988; 5,660,984; and 4,699,717), by reversed phase (e.g. Hirbayashiet al., J. of Chromatog. 722:135-142 (1996); U.S. Pat. No. 5,057,426, byaffinity chromatography (e.g., U.S. Pat. No. 5,712,383; and PolyATract®mRNA Purification System (Promega Corp., Madison, Wis.; see Promega'sTechnical Manual No. TM031), and by matricies which employ a combinationof the above isolation modes (see, e.g. U.S. Pat. No. 5,652,348; J.Chromatography 270:117-126(1983))

One of the first solid phases developed for use in isolating nucleicacids was a specialized resin of porous silica gel particles designedfor use in high performance liquid chromatography (HPLC). The surface ofporous silica gel particles was functionalized with anion-exchangerswhich could exchange with plasmid DNA under certain salt and pHconditions. See, e.g. U.S. Pat. Nos. 4,699,717, and 5,057,426.Machrey-Nagel Co. (Düren, Germany) was one of the first companies toprovide HPLC columns packed with such anion-exchange silica gelparticles, and it continues to sell such columns today. See, e.g.Information about NUCLEOGEN® 4000-7DEAE in product informationdownloaded from the Machrey-Nagel homepage on the Internet on Jun. 12,1998, at http://www.machrey-nagel.com. Each such column was designed sothat plasmid DNA bound thereto is eluted in an aqueous solutioncontaining a high concentration of a highly corrosive salt (e.g. plasmidDNA is eluted from the NUCLEOGEN® 4000-7DEAE column in 6 M urea). Eachsuch column had to be washed thoroughly between each isolation procedureto remove the corrosive salt and contaminants bound to the column withthe DNA from the system. The nucleic acid solution eluted therefrom alsohad to be processed further to remove the corrosive salt therefrombefore it could be used in standard molecular biology techniques, suchas cloning, transformation, digestion with restrictive enzymes, oramplification.

Various silica-based solid phase separation systems have been developedsince the early HPLC systems described above. (See, e.g. the silica geland glass mixture for isolating nucleic acids according to U.S. Pat. No.5,658,548, and the porous support with silane bonded phase used toisolate oligonucleotides according to U.S. Pat. No. 4,767,670.) Modemsilica-based systems utilize controlled pore glass, filters embeddedwith silica particles, silica gel particles, resins comprising silica inthe form of diatomaceous earth, glass fibers or mixtures of the above.Each modem silica-based solid phase separation system is configured toreversibly bind nucleic acid materials when placed in contact with amedium containing such materials in the presence of chaotropic agents.Such solid phases are designed to remain bound to the nucleic acidmaterial while the solid phase is exposed to an external force such ascentrifugation or vacuum filtration to separate the matrix and nucleicacid material bound thereto from the remaining media components. Thenucleic acid material is then eluted from the solid phase by exposingthe solid phase to an elution solution, such as water or an elutionbuffer. Numerous commercial sources offer silica-based resins designedfor use in centrifugation and/or filtration isolation systems. See, e.g.Wizard® DNA purification systems products from Promega Corporation(Madison, Wis., U.S.A.); or the QiaPrep® DNA isolation systems fromQiagen Corp. (Chatsworth, Calif., U.S.A.)

Magnetically responsive particles, formerly used to isolate and purifypolypeptide molecules such as proteins or antibodies, have also beendeveloped for use as solid phases in isolating nucleic acids. Severaldifferent types of magnetically responsive particles designed forisolation of such materials are described in the literature, and many ofthose types of particles are available from commercial sources. Suchparticles generally fall into either of two categories, those designedto reversibly bind nucleic acid materials directly, and those designedto reversibly bind nucleic acid materials through an intermediary. Foran example of particles of the first type, see silica based porousparticles designed to reversibly bind directly to DNA, such as MagneSil™particles from Promega, or BioMag® magnetic particles from PerSeptiveBiosystems. For examples of particles and systems of the second typedesigned to reversibly bind one particular type of nucleic acid (mRNA),see the PolyATract® Series 9600™ mRNA Isolation System from PromegaCorporation (Madison, Wis., U.S.A.); or the ProActive® line ofstreptavidin coated microsphere particles from Bangs Laboratories(Carmel, Ind., U.S.A.). Both of these latter two systems employmagnetically responsive particles with avidin subunits covalentlyattached thereto, and streptavidin with an oligo dT moiety covalentlyattached thereto. The streptavidin-oligo dT molecules act asintermediaries, hybridizing to the poly A tail of mRNA molecules whenplaced into contact therewith, then binding to the particles through areleasable streptavidin-avidin bond.

The indirect binding magnetic separation systems for nucleic acidisolation or separation all require at least three components, i.e.magnetic particles, an intermediary, and a medium containing the nucleicacid material of interest. The intermediary/nucleic acid hybridizationreaction and intermediary/particle binding reaction often requiredifferent solution and/or temperature reaction conditions from oneanother. Each additional component or solution used in the nucleic acidisolation procedure adds to the risk of contamination of the isolatedend product by nucleases, metals, and other deleterious substances.

Various types of magnetically responsive silica based particles havebeen developed for use as solid phases in direct or indirect nucleicacid binding isolation methods. One such particle type is a magneticallyresponsive glass bead, preferably of a controlled pore size. See, e.g.Magnetic Porous Glass (MPG) particles from CPG, Inc. (Lincoln Park,N.J., U.S.A.); or porous magnetic glass particles described in U.S. Pat.Nos. 4,395,271; 4,233,169; or 4,297,337. Nucleic acid material tends tobind very tightly to glass, however, so that it can be difficult toremove once bound thereto. Therefore, elution efficiencies from magneticglass particles tend to be low compared to elution efficiencies fromparticles containing lower amounts of a nucleic acid binding materialsuch as silica

Another type of magnetically responsive particle designed for use as asolid phase in direct binding and isolation of nucleic acids,particularly DNA, is a particle comprised of agarose embedded withsmaller ferromagnetic particles and coated with glass. See, e.g. U.S.Pat. No. 5,395,498. A third type of magnetically responsive particledesigned for direct binding and isolation of nucleic acids is producedby incorporating magnetic materials into the matrix of polymeric silicondioxide compounds. See, e.g. German Patent No. DE 43 07 262 A1. Thelatter two types of magnetic particles, the agarose particle and thepolymeric silicon dioxide matrix, tend to leach iron into a medium underthe conditions required to bind nucleic acid materials directly to eachsuch magnetic particle. It is also difficult to produce such particleswith a sufficiently uniform and concentrated magnetic capacity to ensurerapid and efficient isolation of nucleic acid materials bound thereto.

Silica-based solid phase nucleic acid isolation systems, whethermagnetic or non-magnetic based or configured for direct or indirectbinding, are quick and easy to use and do not require the use ofcorrosive or hazardous chemicals. However, such are ineffective atisolating nucleic acids from contaminants, such as endotoxins, whichtend to bind to and elute from such solid supports under the sameconditions as nucleic acids. See, e.g. Cotten, Matt et al. Gene Therapy(1994) 1:239-246.

Some nucleic isolation systems have been developed in which a nucleicacid solution containing proteins is pre-treated with proteases todigest at least some of the proteins contained therein prior toisolation of the nucleic acid using a silica-based solid support of thetype described above. See, e.g. QiaAmp™ Blood Kit provided by QIAGENInc. (Santa Clarita, Calif.), which utilizes protease; and Wizard® PlusSV Minipreps DNA Purification System provided by Promega Corp. (Madison,Wis.), which utilizes an alkaline protease. However, such pretreatmentsystems require the introduction of one contaminant into a mixture todigest another contaminant. Carry-over proteases can limit the utilityof nucleic acids isolated using such modified silica-based systems atleast as much as nucleic acid samples contaminated with the proteins theproteases are introduced to digest Specifically, given the propersolution conditions, proteases in a nucleic acid solution will digestany proteins introduced into the solution, including enzymes introducedtherein to modify, cut, or transcribe the nucleic acid contained thereinfor downstream processing or analysis. Protease addition, incubation andremoval steps also drive up the cost of nucleic acid isolation, costingtime and money compared to isolation systems with no such additionalsteps.

In all the solid phase systems described above, each solid phase usedtherein has a substantially uniform surface composition designed to bindto a nucleic acid of interest, in the form of a silica or silica gelsurface, or in the form of a silica gel or polymer surface modified withchemical groups exhibiting anion exchanger activities. Bimodal andmultimodal systems have also been developed, such as systems: (1) inwhich multiple columns each of which contains a solid phase modifiedwith a different chemical group from the other columns in the system(e.g., Wheatley J. B., J. Chromatogr. (1992) 603: 273); (2) in which asingle column is used with a single solid phase with at least twodifferent chemical groups (e.g., Patent '680; Little, E. L. et al.,Anal. Chem. (1991) 63: 33); or (3) in which two different solid phasesare employed in the same column, wherein the two solid phases areseparated from one another within the column by solid porous dividers(e.g., U.S. Pat. No. 5,660,984). Each of the chemical groups on thesurface of the solid supports in the single column or multicolumnmultimodal systems is configured to bind to different materials inwhatever substrate is introduced into the system. Only a few suchbimodal or multimodal column chromatography systems have been developedspecifically for nucleic acid isolation (see, e.g. U.S. Pat. No.5,316,680). Surface group combinations used in such solid phase systemsinclude reverse phase, ion exchange, size exclusion, normal phase,hydrophobic interaction, hydrophilic interaction, and affinitychromatography. Such systems are designed such that only one of thesurface groups binds a target species, such as a nucleic acid, while theother surface group(s) bind to and remove one or more non-target speciesin a mixture.

Bimodal and multimodal systems are far from simple, efficientalternatives to conventional organic or resin methods of nucleic acidisolation described above. Multi-column systems are inherently complexto run, as each column requires a unique set of mobile phase conditionsto bind and/or release the desired target or non-target species bound tothe stationary solid phase of the system. Non-target species tend toblock adjacent functional groups configured to bind to the targetspecies, thus adversely affecting overall yield. Also, all the bimodalor multimodal systems are only designed to separate a target speciesfrom other species for which functional groups have affinity.

At least one mixed mode ion exchange solid phase system has beendeveloped for use in isolating certain types of target compounds, suchas proteins or peptides, from an aqueous solution. See U.S. Pat. No.5,652,348 (hereinafter, “Burton et al. '348”) at col. 4, lines 21 to 25.The mixed mode ion exchange system of Burton et al. '348 comprises asolid support matrix with ionizable ligands covalently attached to thesold support matrix. The ionizable ligand is capable of exchanging withand adsorbing the target compound at a first pH and of releasing ordesorbing the target compound at a second pH. The ionizablefunctionality is “either further electrostatically charged or charged ata different polarity at the second pH”. (Burton et al. '348, claim 1,col. 25, lines 46-50). The examples of mixed mode ion exchange solidphase systems provided in the Burton et al. '348 patent contain only asingle ionizable functionality, an amine residue capable of acting as ananion exchange group at the first pH. The concentration of ionizableligands present on the solid support matricies disclosed in Burton etal. '348 is sufficiently high to “permit target protein binding at bothhigh and low ionic strength”. The only ligand density specificallydisclosed and claimed as sufficiently high for the mixed mode ionexchange solid phase of Burton et al. '348 to bind to target proteins athigh and low ionic strength is a ligand density which is “greater thanthe smaller of at least about 1 mmol/gram dryweight of resin or at leastabout 150 μmol/ml of resin” (col 13, lines 22-23; and claim 1). Themixed mode ion exchange system of Burton et al. '348, is specificallydesigned for use in the isolation of proteins and peptides, not nucleicacids or oligonucleotides.

Materials and methods are needed which can quickly, safely, andefficiently isolate target nucleic acids which are sufficiently free ofcontaminants to be used in molecular biology procedures. The presentinvention addresses the need for materials and methods which provide arapid and efficient means for isolating target nucleic acids from anymixture of target nucleic acids and contaminants, including lysates ofgram-negative bacteria, thereby providing purified nucleic acids whichcan be used in a variety of biological applications, includingtransfection of cultured cells and in vivo administration of nucleicacids to organisms.

BRIEF SUMMARY OF THE INVENTION

Briefly, in one aspect, the present invention is a pH dependent ionexchange matrix designed for use in isolating a target nucleic acid byadsorbing to the target nucleic acid at an adsorption pH and byreleasing the target nucleic acid at a desorption pH which is higherthan the adsorption pH.

In one embodiment of the present invention, the pH dependent ionexchange matrix comprises a solid support and a plurality of first ionexchange ligands, wherein each first ion exchange ligand comprises:

a cap comprising an amine with a pK of less than about 9, wherein theamine is selected from the group consisting of a primary, a secondary,or a tertiary amine;

a spacer covalently attached to the cap, the spacer comprising a spaceralkyl chain with an amine terminus, and an acidic moiety covalentlyattached to the spacer alkyl chain; and

a linker comprising a linker alkyl chain covalently attached to thesolid support at a first end of the linker alkyl chain and covalentlyattached to the amine terminus of the spacer at a second end of thelinker alkyl chain.

In another embodiment, the present invention is a bimodal pH dependention exchange matrix having the same basic structure as the matrixdescribed above except that the spacer does not include an acidicmoiety, wherein the bimodal pH dependent ion exchange matrix furthercomprises a plurality of second ion exchange ligands covalently attachedto the solid support. Each second ion exchange ligand comprises an alkylchain with an acidic substituent covalently attached to the alkyl chain.

In another aspect, the present invention is a method of isolating atarget nucleic acid using a pH dependent ion exchange matrix, accordingto steps comprising:

(a) providing the pH dependent ion exchange matrix;

(b) combining the matrix with a mixture comprising the target nucleicacid and at least one contaminant;

(c) incubating the matrix and mixture at an adsorption pH, wherein thetarget nucleic acid adsorbs to the matrix, forming a complex;

(d) separating the complex from the mixture; and

(e) combining the complex with an elution solution at a desorption pH,wherein the target nucleic acid is desorbed from the complex.

In yet another aspect, the present invention is a method of making a pHdependent ion exchange matrix, comprising the steps of:

(a) providing a solid phase;

(b) providing a linker comprising a linker alkyl chain having a firstend and a second end;

(c) combining the solid phase and the linker under conditions where acovalent bond is formed between the first end of the linker alkyl chainand the solid phase, thereby forming a linker-modified solid phase;

(d) providing an alkyl amine comprising:

a cap comprising an amine with a pK of less than about 9, wherein theamine is selected from the group consisting of a primary, secondary, ortertiary amine;

a spacer which is covalently attached to the cap, wherein the spacercomprises a spacer alkyl chain with an amino terminus, and an acidicsubstituent covalently attached to the spacer alkyl chain; and

(e) combining the linker-modified solid phase with the alkyl amine underconditions where a covalent bond is formed between the amino terminus ofthe spacer alkyl chain and the second end of the linker.

In yet another embodiment, the present invention is a method of making apH dependent ion exchange matrix, according to the steps comprising:

(a) providing a solid support;

(b) providing a first ion exchange ligand comprising:

a cap comprising an amine with a pK of less than about 9, wherein theamine is selected from the group consisting of a primary, secondary, ortertiary amine;

a spacer covalently attached to the cap, the spacer comprising a spaceralkyl chain with an amine terminus, an acidic substitutent covalentlyattached to the spacer alkyl chain, and a protecting group covalentlyattached to the acidic substituent; and

a linker comprising a linker alkyl chain having a first end and a secondend, wherein the second end is covalently attached to the amine terminusof the spacer,

(c) combining the solid phase and the first ion exchange ligand underconditions where a covalent bond is formed between solid phase and thefirst end of the linker alkyl chain; and

(d) deprotecting the acidic substituent of the first ligand.

Another embodiment of the present invention is a method of making abimodal pH dependent ion exchange matrix according to the stepscomprising:

(a) providing a solid support;

(b) providing a first ion exchange ligand comprising:

a cap comprising an amine having a pK of less than about 9, wherein theamine is selected from the group consisting of a primary, secondary, ortertiary amine;

a spacer covalently attached to the cap, the spacer comprising a spaceralkyl chain with an amine terminus; and

a linker comprising a linker alkyl chain having a first end and a secondend, wherein the second end is covalently attached to the amine terminusof the spacer; and

(c) combining the solid phase and the first ion exchange ligand underconditions where a covalent bond is formed between solid phase and thefirst end of the linker alkyl chain.

(d) combining the first ion exchange-modified solid phase with a secondion exchange ligand under conditions where a covalent bond is formedbetween the solid phase and one end of the second ion exchange ligand,wherein the ion exchange ligand comprises a second alkyl chain, anacidic substituent covalently attached to the second alkyl chain, and aprotecting group attached to the acidic substitutent.

(e) removing the protecting group from the acidic substituent.

The methods and materials of the present invention can be used toisolate target nucleic acids including, but not limited to plasmid DNA,total RNA, amplified nucleic acids, and genomic DNA from a variety ofcontaminants, including but not limited to agarose and components of abacteria, animal tissue, blood cells, and non-target nucleic acids.

Applications of the methods and compositions of the present invention toisolate nucleic acids from a variety of different media will becomeapparent from the detailed description of the invention below. Thoseskilled in the art of this invention will appreciate that the detaileddescription of the invention is meant to be exemplary only and shouldnot be viewed as limiting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a method of making a pH dependent ion exchange matrixwherein a cap, comprising an amine with a pK of less than about 9, iscovalently attached to a solid phase through a glycidyl linker.

FIG. 2 illustrates a method of making a pH dependent ion exchange matrixby linking an amino alkyl spacer and a cap comprising an aromatichydrocarbon ring with an amine member, to a sold phase through a urealinkage.

FIG. 3 illustrates a method of making a bimodal pH dependent ionexchange matrix.

FIG. 4 is a reproduction of a photograph of amplified DNA isolated withMagnasil™ and with pH dependent silica magnetic particles, as describedin Example 12, then fractionated by gel electrophoresis, and stainedwith ethidium bromide.

DETAILED DESCRIPTION OF THE INVENTION

The term “alkyl chain” as used herein refers to a straight chain alkaneoptionally substituted with at least one oxygen, nitrogen, or sulfuratom.

The term “pH dependent ion exchange matrix”, as used herein, refers to amatrix of a solid support and a plurality of ligands covalently attachedthereto wherein at least one ligand includes an acidic moiety, and thesame or a different ligand covalently attached to the same matrixcomprises an amine with a pK of less than about 9, wherein the matrixhas a capacity to adsorb to a target nucleic acid at a first pH and todesorb the target nucleic acid at a desorption pH which is higher thanthe first pH.

The term “solid phase” is used herein in a standard chromatographicsense, to refer to an insoluble, usually rigid, matrix or stationaryphase which interacts with a solute, in this case a target nucleic acid,in a solute mixture. The term solid phase, as used herein, specificallyincludes stationary phases in liquid chromatography (LC), high pressureliquid chromatography (HPLC), particulate matrices embedded into orbound to filters, and magnetic or non-magnetic porous matrix particleswhich interact with solutes when added directly to a solute mixture.

The term “silica gel” as used herein refers to chromatography gradesilica gel, a substance which is commercially available from a number ofdifferent sources. Silica gel is most commonly prepared by acidifying asolution containing silicate, e.g. by acidifying sodium silicate to a pHof less than 11, and then allowing the acidified solution to gel. See,e.g. silica preparation discussion in Kurt-Othmer Encyclopedia ofChemical Technology, Vol. 21, 4th ed., Mary Howe-Grant, ed., John Wiley& Sons, pub., 1997, p. 1021.

The term “glass particles” as used herein means particles of crystallineor vitreous silicas, even though crystalline silicas are not formally“glasses” because they are not amorphous, or particles of glass madeprimarily of silica. The term includes quartz, vitreous silica,controlled pore glass particles, and glass fibers.

As used herein, the term “silica magnetic particles” refers to silicabased solid phases which are further comprised of materials which haveno magnetic field but which form a magnetic dipole when exposed to amagnetic field, i.e., materials capable of being magnetized in thepresence of a magnetic field but which are not themselves magnetic inthe absence of such a field.

The term “magnetic” as used to refer to silica magnetic particlesincludes materials which are paramagnetic or superparamagneticmaterials. The term “magnetic”, as used herein, also encompassestemporarily magnetic materials, such as ferromagnetic or ferromagneticmaterials. Except where indicated otherwise below, the silica magneticparticles used in this invention preferably comprise a superparamagneticcore coated with siliceous oxide, having a hydrous siliceous oxideadsorptive surface (i.e. a surface characterized by the presence ofsilanol groups).

The term “surface”, as used herein, refers to the portion of the supportmaterial of a solid phase which comes into direct contact with asolution when the solid phase is combined therewith.

The term “nucleic acid” as used herein refers to any DNA or RNA moleculeor a DNA/RNA hybrid molecule. The term includes plasmid DNA, amplifiedDNA or RNA fragments, total RNA, mRNA, and genomic DNA.

The term “target nucleic acid” as used herein refers to the particularspecies of nucleic acid to be isolated in any particular application ofthe methods or use of the pH dependent ion exchange matrix of thepresent invention. The target nucleic acid is preferably at least 20nucleotides long, more preferably at least 100 nucleotides long, andmost preferably at least 1,000 nucleotides long.

The solid support component of the pH dependent ion exchange matrix canbe made of any common support material, including soft gel supports suchas agarose, polyacrylamide, or cellulose, or hard support material suchas polystyrene, latex methacrylate, or silica When the solid phasesupport material is silica, it is preferably in the form of silica gel,siliceous oxide, solid silica such as glass or diatomaceous earth, or amixture of two or more of the above. Silica based solid phases suitablefor use in the pH dependent ion exchange matrixes of the presentinvention include the mixture of silica gel and glass described in U.S.Pat. No. 5,658,548, the silica magnetic particles described in PCTPublication Number WO 98/31840, and solid phases sold by PromegaCorporation for use in plasmid DNA isolation, i.e. Wizard® Minipreps DNAPurification Resin. Silica gel particles are particularly preferred foruse as the solid phase in the pH dependent ion exchange matrix andmethods of the present invention. Silica gel particles are stable atmuch higher pressures than solid phases made from soft gel supportmaterial, making the silica gel solid phases suitable for HPLC as wellas LC and batch separation applications.

The pH dependent ion exchange matrix used in the present invention ispreferably in a form which can be separated from a solute mixturecomprising the target nucleic acid and at least one contaminant afterthe solute mixture is combined therewith, by application of an externalforce. A skilled artisan would appreciate that the type of externalforce suitable for use in separating the matrix from the solute mixdepends upon the form in which the matrix is presented to the solutemix, and upon the physical properties of the matrix itself. For example,gravity can be used to separate the pH dependent ion exchange matrixfrom the solute mix when the matrix is in the form of a chromatographicresin loaded on an LC column, when the matrix is in the form of silicaparticles (e.g., controlled pore glass, silica gel particles, or silicamagnetic particles) which are added batch-wise to a solute mixture andthen separated therefrom by decantation or filtration, or when themixed-mode matrix is in the form of a filter with silica particles orchromatographic resin embedded into or attached thereto.

The external force used in the method of isolation is high pressureliquid when the pH dependent ion exchange matrix is the stationary phaseof a high pressure liquid chromatography column (HPLC). Other forms ofexternal force suitable for use in the method of this invention includevacuum filtration (e.g. when the solid phase component of the matrix isparticles of controlled pore glass, particles of silica gel or silicamagnetic particles, or mixtures of one or more of the above types ofparticles embedded into or attached to a filter), centrifugation (e.g.when the mixed-bed solid phase is particulate), or magnetic (e.g. whenthe mixed-bed solid phase comprises magnetic or paramagnetic particles).

When the solid phase component of the pH dependent ion exchange matrixis a silica gel particle, it is most preferably a silica magneticparticle. A silica magnetic particle can be separated from a solutionusing any of the external means described above for use with other typesof solid phases, such as those described above. However, unlike theother solid phases, a silica magnetic particle can be separated from asolution by magnetic force, a quick and efficient means of separating amatrix from a solution.

When the solid support component of the pH dependent ion exchange matrixis a silica magnetic particle, the size of the particle is preferablyselected as follows. Smaller silica magnetic particles provide moresurface area (on a per weight unit basis) for covalent attachment to theplurality of ion exchange ligands, but smaller particles are limited inthe amount of magnetic material which can be incorporated into suchparticles compared to larger particles. The median particle size of thesilica magnetic particles used in a particularly preferred embodiment ofthe present invention is about 1 to 15 μm, more preferably about 3 to 10μm, and most preferably about 4 to 7 μm. The particle size distributionmay also be varied. However, a relatively narrow monodal particle sizedistribution is preferred. The monodal particle size distribution ispreferably such that about 80% by weight of the particles are within a10 μm range of the median particle size, more preferably within an 8 μmrange, and most preferably within a 6 μm range.

The solid support component of the pH dependent ion exchange matrix canbe porous or non-porous. When the solid support is porous, the pores arepreferably of a controlled size range sufficiently large to admit thetarget nucleic acid material into the interior of the solid phaseparticle, and to bind to functional groups or silica on the interiorsurface of the pores. The total pore volume of a silica magneticparticle, as measured by nitrogen BET method, is preferably at leastabout 0.2 ml/g of particle mass. The total pore volume of porous silicamagnetic particles particularly preferred for use as components of thepH dependent ion exchange matrix of the present invention, as measuredby nitrogen BET, is preferably at least about 50% of the pore volume iscontained in pores having a diameter of 600 Å or greater.

Silica magnetic particles may contain substances, such as transitionmetals or volatile organics, which could adversely affect the utility oftarget nucleic acids substantially contaminated with such substances.Specifically, such contaminants could affect downstream processing,analysis, and/or use of the such materials, for example, by inhibitingenzyme activity or nicking or degrading the target nucleic acidsisolated therewith. Any such substances present in the silica magneticparticles used in the present invention are preferably present in a formwhich does not readily leach out of the particle and into the isolatedbiological target material produced according to the methods of thepresent invention. Iron is one such undesirable at least onecontaminant, particularly when the biological target material is atarget nucleic acid.

Iron, in the form of magnetite, is present at the core of particularlypreferred forms of silica magnetic particles used as the solid phasecomponent of the pH dependent ion exchange matrixes of the presentinvention. Iron has a broad absorption peak between 260 and 270nanometers (nm). Target nucleic acids have a peak absorption at about260 μm, so iron contamination in a target nucleic acid sample canadversely affect the accuracy of the results of quantitativespectrophotometric analysis of such samples. Any iron containing silicamagnetic particles used to isolate target nucleic acids using thepresent invention preferably do not produce isolated target nucleic acidmaterial sufficiently contaminated with iron for the iron to interferewith spectrophotometric analysis of the material at or around 260 nm.

The most preferred silica magnetic particles used in the matrixes andmethods of the present invention, siliceous oxide coated silica magneticparticles, leach no more than 50 ppm, more preferably no more than 10ppm, and most preferably no more than 5 ppm of transition metals whenassayed as follows. Specifically, the particles are assayed as follows:0.33 g of the particles (oven dried @ 110° C.) are combined with 20 ml.of 1N HCl aqueous solution (using deionized water). The resultingmixture is then agitated only to disperse the particles. After about 15minutes total contact time, a portion of the liquid from the mixture isthen analyzed for metals content. Any conventional elemental analysistechnique may be employed to quantify the amount of transition metal inthe resulting liquid, but inductively coupled plasma spectroscopy (ICP)is preferred.

At least two commercial silica magnetic particles are particularlypreferred for use in the matrix of the present invention, BioMag®Magnetic Particles from PerSeptive Biosystems, and the MagneSil™Particles available from Promega Corporation (Madison, Wis.). Any sourceof magnetic force sufficiently strong to separate the silica magneticparticles from a solution would be suitable for use in the nucleic acidisolation methods of the present invention. However, the magnetic forceis preferably provided in the form of a magnetic separation stand, suchas one of the MagneSphere® Technology Magnetic Separation Stands (cat.no.'s Z5331 to 3, or Z5341 to 3) from Promega Corporation.

The pH dependent ion exchange matrices of the present invention allinclude a plurality of first ion exchange ligands covalently attached toa solid phase, according the general structure of formula (I), below:

wherein the wavy line represents a surface of the solid phase. LINKERcomprises a linker alkyl chain, preferably an alkyl chain which includesthree (3) to eight (8) carbon atoms. The LINKER preferably also includesat least one additional member selected from the group consisting ofoxygen, amine, and carbonyl. The LINKER is preferably an epoxide, suchas a glycidyl moiety, or a urea linkage. The SPACER comprises a spaceralkyl chain with an amine terminus, wherein the amine terminus iscovalently attached to the LINKER. The other end of the spacer alkylchain is covalently attached to the CAP. The SPACER alkyl chain can besubstituted by at least one sulphur residue. The CAP comprises aprimary, secondary, or tertiary amine with a pK value less than 9. TheCAP preferably further comprises an aromatic hydrocarbon ring, whereinthe amine is either attached to or a member of the ring. When the CAPcomprises an aromatic hydrocarbon ring and an amine, the amine ispreferably a member of the ring. The CAP more preferably comprises afive or six member aromatic amine ring, such as imidazole or pyridine.

In one embodiment of the present invention, wherein the plurality offirst ion exchange ligands are the only ion exchange ligands attached tothe solid phase, the SPACER further comprises a first acidic moietycovalently attached to the spacer alkyl chain. The acidic moiety ispreferably a carboxyl residue. In this embodiment of the invention, atleast one basic (the amine member of the aromatic hydrocarbon) and atleast one acidic moiety are both members of the first ligand. The SPACERis preferably selected from the group consisting of cysteine, alanine,and the alkyl chain portion of a polar amino acid consisting of an alkylchain and an aromatic hydrocarbon such as histamine and histidine.SPACER and CAP together most preferably define a histamine or ahistidine moiety.

In another embodiment, the present invention is a pH dependent ionexchange matrix comprising a plurality of first ion exchange ligands anda plurality of second ion exchange ligands covalently attached to thesame solid support, such as the same silica magnetic particle. Thesecond ion exchange ligand comprises a second alkyl chain and an ionexchange residue covalently attached thereto. The second alkyl chain ispreferably an unbranched alkane of one (1) to five (5) carbon atoms. Theion exchange residue is preferably an acidic moiety, more preferably acarboxylic acid. The second ion exchange ligand is most preferablypropionate.

In this second embodiment of the pH dependent ion exchange matrix, eachfirst ion exchange ligand can have the same structure as set forth inFormula (I), above, except that the first ion exchange ligand need nothave an acidic moiety covalently attached to the spacer alkyl chain whenthe second ion exchange ligand includes such a moiety. When the secondion exchange ligand includes an acidic moiety, it is preferably acarboxylic acid residue, more preferably a carboxylic acid residuecovalently attached to the terminus of the second alkyl chain.

The second type of pH ion exchange matrix described immediately above,hereinafter the “bimodal” ion exchange matrix, preferably has an acidicmoiety on one ligand, the second ion exchange ligand, and at least onebasic moiety on the other ligand, the amine member of the aromatichydrocarbon ring component of the first ion exchange ligand. In thatpreferred configuration, target nucleic acid binding and releasecapacity of the matrix can be controlled and even fine tuned by varyingthe relative proportion of first and second ion exchange ligandscovalently bound to the solid support. This feature of the bimodal ionexchange matrix makes it particularly desirable for use in the methodsof the present invention, although the monomodal ion exchange matrixdescribed above is also well suited for use in the isolation of targetnucleic acids according to the present methods. When the solid phase issilica based, each ion exchange ligand is preferably covalently attachedto the solid phase through a silane group, as shown in formula (II),below:

Wherein, R¹ is selected from the group consisting of —OH, —OCH₃, and—OCH₂CH₃; and R² is represented by the formula —(OSiR¹ ₂)_(y)—R¹,wherein y is at least 0. When y is zero (0), the ligand is connected tothe solid support through a silane monomer. When y is greater than zero,the connection is through a silane polymer.

Target nucleic acids are inherently negatively charged at any pH higherthan 2, and can, therefore, reversibly bind to anion-exchangers undersolution conditions where ions can be exchanged between theanion-exchanger and the target nucleic acid. The pH dependent ionexchange matrix of the present invention is an anion exchanger at afirst pH in which the matrix present is neutral to positively charged.At a second, higher pH the matrix becomes neutral to negatively chargeddepending on the pK of the acidic moiety of the ion exchange ligand. Thetarget nucleic acid can adsorb to the matrix at the first pH and desorbfrom the matrix at the second pH. The possible pH range for each of thefirst and second pH depends upon the nature of the plurality of ionexchange ligands component of the pH dependent ion exchange matrix.

The plurality of ligands include at least one anion-exchange moiety andat least one cation-exchange moiety. The at least one anion-exchangemoiety of the pH dependent ion exchange matrix is at least one aminewith a pK of less than 9, wherein the amine is selected from the groupconsisting of a primary, secondary, or tertiary amine. The at least onecation-exchange moiety is an acidic moiety, preferably selected from thegroup consisting of hydroxyl and carboxyl.

The pH dependent ion exchange solid phase of the present invention isdesigned for use in the isolation of target nucleic acids. Both theligand configuration, described above, and ligand density can beadjusted to ensure optimal adsorption and desorption of a given targetnucleic acid. The highest ligand density suitable for use in thematrices of the present invention is 500 μmol per gram of dry weight.The lowest ligand density suitable for use in the pH dependent ionexchange matrices of the present invention is about 25 μmol/g dryweight. The ligand density in the matrices of the present invention ismost preferably between 50 and 200 μmol/g dry weight of solid phase.

The anion exchange moiety and cation exchange moiety of the presentmatrix vary in charge depending upon solution conditions. In thepresence of a solution having a first pH, the basic moiety (i.e., theamine) is positively charged and the matrix is capable of exchangingwith the target nucleic acid. In the presence of a solution having asecond pH which is higher than the first pH, the acidic moiety has anegative charge and the basic moiety has a neutral charge. The matrix isdesigned to adsorb the target nucleic acid at the first pH and to desorbthe target nucleic acid at a pH which is at least about the second pH.pH conditions necessary to ensure adsorption and desorption of thetarget nucleic acid to the matrix of the present invention depend uponthe salt conditions of the adsorption and desorption solutions, and uponthe specific composition and density of the plurality of ligandsattached to the solid phase. Specifically, the first pH, at whichdesorption takes place, is preferably between pH 6 and 8 when the ionicstrength of the solution is preferably no higher than about 1 M salt,more preferably no higher than about 500 mM salt, and most preferably nohigher than about 50 mM salt.

The method of isolating a target nucleic acid of the present inventioncan employ either type of pH dependent ion exchange matrix of thepresent invention described above alone, or a mixed bed of a pHdependent ion exchange matrix and another type of matrix capable ofbinding and releasing the target nucleic acid under a different set ofsolution conditions such as is described in the U.S. patent applicationSer. No. 09/312,139, filed May 14, 1999 for MIXED BED SOLID PHASE ANDITS USE IN THE ISOLATION OF NUCLEIC ACIDS.

The present method comprises the steps of providing the pH dependent ionexchange matrix to be used in the method, providing a mixture comprisingthe target nucleic acid and at least one contaminant, combining themixture and the matrix at a first pH under conditions where the targetnucleic acid adsorbs to the matrix to form a complex, separating thecomplex from the mixture, and desorbing the target nucleic acid from thecomplex by combining the complex with an elution solution at adesorption pH. The exact solution conditions necessary to ensureadsorption and desorption of the target nucleic acid to the matrix varydepending upon several factors, including the nature of the targetnucleic acid (e.g., RNA or DNA, molecular weight, and nucleotidesequence composition), the pKa and pKb of the acidic and basic subunitsof the ligands, ligand density on the surface of a solid phase, andcapacity of the solid phase to bind directly to the target nucleic acid.Some contaminants in the mixture can also interfere with adherence tothe matrix.

Preferably, no chaotropic agent (e.g. guanidine hydrochloride orguanidine isothiocyanate) or low molecular weight alcohol (e.g. ethanolor methanol) is included in any of the solutions which come into contactwith the matrix regardless of the particular species of target nucleicacid. Even trace amounts of chaotropic agents or ethanol in a solutionof target nucleic acid can severely limit the utility of the nucleicacid in downstream processing or analysis.

When the target nucleic acid is plasmid DNA, the pH dependent ionexchange matrix of the present invention can be added directly to thecleared lysate of bacteria transformed with the plasmid DNA and lysedwith an alkaline lysis solution. Alkaline lysis procedures suitable foruse in the present invention can be found in Sambrook et al, MolecularCloning, Vol. 1, 2^(nd) ed. (pub. 1989 by Cold Spring Harbor LaboratoryPress), pp. 1.25-1.28, and in Technical Bulletin Nos. 202, 225, and 259(Promega Corp.). Plasmid DNA from a lysate solution prepared asdescribed above will adsorb to the pH dependent ion exchange matrix uponcombination therewith, provided the overall charge of the matrix ispositive, and provided the charge density is sufficiently high to enableto plasmid DNA to participate in anion exchange with the ion exchangeligands of the matrix at a first pH. Once adsorbed to the matrix to forma complex, the complex can be washed in a wash solution with buffer andsalt solution conditions designed to ensure the plasmid DNA remainsadsorbed to the matrix throughout any such washing steps, while removingat least one contaminant. Finally, the plasmid DNA is eluted from thecomplex by combining the complex with an elution buffer having a secondpH above that of the lysate and wash solutions, wherein the second pH issufficiently high to promote desorption of the plasmid DNA from thematrix.

The pH dependent ion exchange matrix and methods of the presentinvention can be used to isolate genomic DNA from living tissue,including but not limited to blood, semen, vaginal cells, hair, buccaltissue, saliva, tissue culture cells, plant cells, placental cells, orfetal cells present in amniotic fluid and mixtures of body fluids. Whenthe target nucleic acid is genomic DNA, it is necessary to disrupt thetissue to release the target genomic DNA from association with othermaterial in the tissue, so the target genomic DNA can adhere to the pHdependent ion exchange matrix in the presence of a solution at the firstpH. The resulting complex of matrix and genomic DNA is separated fromthe disrupted tissue, and washed to remove additional contaminants (ifnecessary). The genomic DNA is then eluted from the complex by combiningthe complex with an elution solution having a second pH which is higherthan the first pH.

When the target nucleic acid is RNA, adsorption of the target nucleicacid to the pH dependent ion exchange matrix is preferably carried outunder conditions designed to promote preferential adsorption of RNA tothe matrix. When both RNA and DNA are present in a solution, thesolution conditions can be designed to promote preferential adsorptionof RNA to the pH dependent ion exchange matrix. The specific solutionconditions required to preferentially promote adsorption and desorptionof RNA to a pH dependent ion exchange matrix will depend upon thecharacteristics of the matrix itself, and must therefore be determinedfor each matrix.

FIGS. 1 through 3 illustrate three additional embodiments of the presentinvention, specifically, three different methods of making threedifferent types of pH dependent ion exchange matrices. The first suchmethod, one illustrated in FIG. 1, is a method of making a pH dependention exchange matrix by linking a cap, comprising of an aromatichydrocarbon ring with an amine member, wherein the amine has a pK ofless than about 9, to a solid phase through a glycidyl linker. Themethod comprises three steps. In the first step, the compound of formula(IV), a glycidypropylsilane with three identical subunits (“R¹”, whichis —OH, —OCH₃, or —OCH₂CH₃) covalently attached to the silane residue,is combined with a solid phase with at least one surface as shown informula (III), with hydroxyl groups covalently attached thereto, underconditions designed to promote the formation of a covalent bond betweenthe silane residue, forming the glycidyl-modified solid phase of formula(V). Finally, the glycidyl modified solid phase is combined with eitheran amino acid which includes an amino acid with an aromatic hydrocarbonring with an amine member, such as histidine, or a amino acid covalentlyattached to an aromatic hydrocarbon, such as pyridyl-cysteine orpyridyl-alanine, under conditions designed to promote formation of apeptide bond between the two compounds through the N-terminus of theamino acid or amino acid moiety. Preferred compounds used in thisparticular step of the method are represented as R²H, wherein thestructures for the R² component of each such compound (i.e., histidine,pyridyl-cysteine, and pyridyl-alanine), are illustrated in FIG. 1. Theend product of this reaction is the pH dependent ion exchange matrix offormula (VI).

The present invention is also a method of making a pH dependent ionexchange matrix by linking a first moiety, comprising an amino alkylspacer and a cap comprising an aromatic hydrocarbon ring with an aminemember, to a solid phase through a urea linkage. FIG. 2 illustrates sucha method of synthesis wherein histidine is the first moiety linked tothe solid phase. However, it is contemplated that substantially the sameprocedure could be used to link other moieties to solid phases throughurea, including histamine. As illustrated in FIG. 2, histidine modifiedby protection of the carboxyl residue with a methyl group, according toformula (VII), is combined with the compound of formula (VIII), a3-isocyanto propylsilane with three identical subunits (“R¹”, which is—OH, —OCH₃, or —OCH₂CH₃) covalently attached to the silane residue. Theresulting mixture is allowed to react under conditions which promoteformation of a covalent bond between the N-terminus of the protectedamino acid (histidine protected by a methyl group, in this case) and thecyanato carbon residue of the compound of formula (VIII), resulting inthe formation of a urea residue. The product of the first reaction isthen combined with the solid phase of formula (III) under conditionsdesigned to promote formation of a covalent bond between the silaneresidue of the product and the hydroxyl groups at a surface of the solidphase. The end product of the second reaction is represented by formula(IX). Finally, the protecting group on the carboxylic acid residue ofthe amino acid moiety is removed by reaction with an oxidant, such ashydrochloric acid. The product of the reaction is represented by formula(X).

The present invention is also a method of making a bimodal or multimodalpH dependent ion exchange matrix. FIG. 3 illustrates the synthesis ofone such bimodal matrix, according to the method of the presentinvention. The first step of the method shown in FIG. 3 is the additionof the compound of formula (XI), an imidazole-ethyl-N′-3-propylsilyureawherein three subunits, two R¹ subunits each defined as —OH, —OCH₃, or—OCH₂CH₃ and one R² subunit, defined by the formula —(OSiR¹ ₂)_(y)—R¹wherein y is at least 0. covalently attached to the silane residue, to asolid phase with hydroxyl groups covalently attached thereto, as shownin formula (III). The compound of formula (XI) can be synthesized fromhistidine and 3-isocyanatopropyltri-substituted silane, using a similarprocedure to that used to form the urea linkage in the first step of themethod discussed immediately above. The compound of formula (XI) and thesolid phase of formula (III) are allowed to react under conditionsdesigned to promote formation of a covalent bond between the silaneresidue of the compound of formula (XI) and the hydroxyl groups at thesurface of the solid phase, thereby forming the solid phase with a firsttype of linker attached thereto, the structure of formula (XII).

The synthesis of the bimodal and multimodal pH dependent ion exchangematricies continues with the addition of at least one other linker. In abimodal matrix, the at least one other linker is a second linker whichincludes an acidic group covalently attached thereto. Attachment of asecond linker to the structure of formula (XII) according to the presentmethod is illustrated in FIG. 3. An alkyl chain with a protected acidicgroup covalently attached thereto and a terminal silane residue withthree identical subunits (“R³”, which is —OH, —OCH₃, or —OCH₂CH₃)covalently attached to the silane residue, such as the compound offormula (XIII), is combined with the solid phase/first linker compoundof formula (XII) under conditions which promote the formation of acovalent bond between the silane residue and the hydroxyl groups at asurface of the solid phase. The protecting group (e.g., a methylresidue) is then removed from the resulting compound of formula (XIV),using an oxidant such as HCl, thereby forming the compound of formula(XV). The silane residue of both the intermediate compound formula (XIV)and the end product of formula (XV) has two subunits attached thereto,R³ and R⁴, wherein R³ is —OH, —OCH₃, or —OCH₂CH₃, and R⁴ is —OSiR³₂)_(z)—R³, wherein z is at least 0.

Multimodal pH dependent ion exchange matrices can also be made, bycovalently attaching additional linkers with acidic or basic residues toa solid phase to fine tune the charge density and overall charge of asolid phase to select for particular target nucleic acids.

The following, non-limiting examples teach various embodiments of theinvention. In the examples, and elsewhere in the specification andclaims, volumes and concentrations are at room temperature unlessspecified otherwise. The magnetic silica particles used in the examplesbelow were all either porous or nonporous MagneSil™ particles having thegeneral preferred dimensions and siliceous oxide coating described aspreferred above. More specifically, the porous MagneSil™ particles usedin the Examples below were taken from either of two batches of particleshaving the following characteristics: (1) a surface area of 55 m²/g,pore volume of 0.181 ml/g for particles of <600 Å diameter, pore volumeof 0.163 ml/g for particles of >600 Å diameter, median particle size of5.3 μm, and iron leach of 2.8 ppm when assayed as described herein aboveusing ICP; or (2) a surface area of 49 m²/g, pore volume of 0.160 ml/g(<600 Å diameter), pore volume of 0.163 ml/g (>600 Å diameter), medianparticle size of 5.5 μm, and iron leach of 2.0 ppm. Specifications ofglass particles used in the examples below are provided below.

One skilled in the art of the present invention will be able to use theteachings of the present disclosure to select and use solid supportsother than the three silica based solid supports used to make the pHdependent ion exchange particles whose synthesis and use is illustratedin the Examples below. The Examples should not be construed as limitingthe scope of the present invention. Other pH dependent ion exchangematrixes, and methods of using the matrixes to isolate target materialaccording to the present invention will be apparent to those skilled inthe art of chromatographic separations and molecular biology.

EXAMPLES

The following examples are given to illustrate various aspects of theinvention, without limiting the scope thereof:

Example 1 Gel Electrophoresis

Samples of target nucleic acids isolated according to proceduresdescribed in Examples below were analyzed for contamination withnon-target nucleic acids, and for size as follows. The samples werefractionated on an agarose gel of appropriate density (e.g., a 1.0%agarose gel was used to analyze plasmid DNA, while a 1.5% agarose gelwas used to analyze RNA). The fractionated nucleic acid was visualizedusing a fluorescent label or by dying the gel with a DNA sensitivestain, such as ethidium bromide or silver staining. The resultingfractionated, visualized nucleic acid was either photographed orvisualized using a fluorimager and the resulting image printed out usinga laser printer.

In some cases, size standards were fractionated on the same gel as thetarget nucleic acid, and used to determine the approximate size of thetarget nucleic acid. In every case where a gel assay was done, thephotograph or fluorimage of the fractionated nucleic acid was inspectedfor contamination by non-target nucleic acids. For example, images offractionated samples of plasmid DNA were inspected for RNA, which runsconsiderably faster than DNA on the same gel, and for chromosomal DNA,which runs considerably slower than plasmid DNA on the same gel. Imagesof isolated plasmid DNA were also inspected to determine whether most ofthe plasmid DNA shown in the image is intact, supercoiled plasmid DNA.

Example 2 Absorption Spectrophotometry

Samples of target nucleic acids isolated from various media, asdescribed below, were also analyzed using absorption spectrophotometry.Absorption measurements were taken at wavelengths of 260, 280, and 230nanometers (nm). A₂₆₀/A₂₈₀ absorption ratios were computed from themeasurements. An A₂₆₀/A₂₈₀ of greater than or equal to 1.80 wasinterpreted to indicate the sample analyzed therein was relatively freeof protein contamination. The concentration of nucleic acid in eachsample was determined from the absorption reading at 260 nm (A₂₆₀).

Example 3 Synthesis of Porous Silica Magnetic pH Dependent Ion ExchangeParticles

Various pH dependent ion exchange ligands were attached to porous silicamagnetic particles, according to the following procedures. The silicamagnetic pH dependent ion exchange particles synthesized as describedherein were used to isolate target nucleic acids, as described insubsequent Examples, below.

A. Preparation of Glycidyl Modified Silica Magnetic Particles

1. Silica magnetic particles were activated by heating under vacuum at110° C. overnight.

2. 10 g of the activated particles were suspended in 100 ml of toluenein a flask, and 3.2 ml of 3-glycidylpropyl-trimethoxysilane was addedthereto.

3. The flask containing the mixture was fitted with a condenser and thereaction was refluxed for 5 hr. After cooling to room temperature, thereaction mixture sat for 48 hr at room temperature.

4. The reaction mixture was then filtered and the retentate, includingglycidyl-modified silica magnetic particles produced in the refluxreaction, were washed with toluene (2×100 ml), hexanes (2×100 ml) andethyl ether (1×150 ml). The washed product was then left to dry in theair.

5. A small portion of the product was further dried in a 110° C. ovenand submitted for elemental analysis. The results (% C 0.75; % H 0.58)are consistent with glycidyl modification of silica gel particles, asillustrated in Formula (III), below. The wavy line in this and otherformulae depicted herein and in the remaining Examples below representsthe surface of a solid phase, a porous silica magnetic particle in thisparticular Example.

wherein, R is —OH, OCH₃, or —OCH₂CH₃.

6. The glycidyl-modified silica magnetic particles produced as describedabove were then further modified by the linkage of an amino acid, suchas histidine, alanine, or cysteine to the particles, by reaction withthe terminal ring of the glycidyl moiety, as described below.

B. Synthesis of Glycidyl-Histidine Modified Silica Magnetic Particles

1. 2.0 g. of D,L-histidine was dissolved in a mixture of 20 ml oftetrahydrofuran and 20 ml of water by heating the solution to reflux.

2. To this solution, 2 g of glycidyl-modified silica magnetic particleswas added and the resulting suspension was refluxed overnight (18 hr).

3. After cooling to room temperature the reaction mixture was filtered,and the retentate, which included glycidyl-histidine modified silicamagnetic particles, was washed once with 100 ml of acetone, three timeswith 150 ml of water, and once with 150 ml of ether. The solid was airdried.

4. A small portion of the dried solid from step 3 was further dried at110° C. and submitted for elemental analysis. Results: % C 1.35; % H0.68; % N 0.50. This results are consistent with glycidyl-histidinelinkage, such as is as shown in Figure (XVII), below:

wherein, R is —OH, OCH₃, or —OCH₂CH₃.

C. Synthesis of Glycidyl -Alanine Modified Silica Magnetic Particles

1. 3-(3-pyridyl)-D-alanine (1 g) was dissolved in 20 ml of water.

2. To this solution 2 g. of glycidyl-modified silica magnetic particleswere added, and the resulting mixture was refluxed overnight.

3. After cooling, the reaction mixture was filtered and washed twicewith water, and once with ethyl ether.

4. Elemental analysis of a sample of the product from step 3 showed: % C0.98; % H 0.56; % N 0.20. This result is consistent withglycidyl-alanine modification, as illustrated in formula (XVIII), below:

wherein, R is —OH, OCH₃, or —OCH₂CH₃.

D. Synthesis of Glycidyl -Cysteine Modified Silica Magnetic Particles

1. 1 g of S-[2-(4-Pyridyl)ethyl]-L-cysteine was suspended in 20 ml ofwater, and heated to dissolve the material.

2. To this solution 2.5 g of glycidyl-modified silica magnetic particleswere added, and the resulting mixture was refluxed overnight.

3. After cooling the reaction mixture was filtered and washed threetimes with water and ethyl ether. The material was air dried.

4. Elemental analysis of the material from step 3 showed: % C 1.08; % H0.42; % N 0.16. This results are consistent with glycidyl-cysteinemodification of silica magnetic particles, as according to formula(XIX), below:

wherein, R is —OH, —OCH₃, or —OCH₂CH₃.

Example 4 Synthesis of Non-Porous Magnesil, Glass Fiber, and Silica GelGlycidyl-Linked pH Dependent Ion Exchange Solid Phases A. Synthesis ofGlycidyl-Histidine Modified Non-Porous Silica Magnetic

1. Glycidyl Modification: 6 ml of non-porous silica magnetic particles(Part No. SMR22-552, provided by W. R. Grace) were suspended in 6 ml oftoluene, and 0.7 ml of 3-Glycidylpropyltrimethoxysilan was added to thesuspension. The resulting mixture was placed on a roto-evaporator andallowed to react overnight. The reaction mixture was filtered and theretentate, including the modified silica magnetic particle product, waswashed once with 20 ml of methylene chloride and once with 20 ml ofethyl ether. The product was dried under vacuum in a desiccator overphosphorous pentoxide. Elemental analysis showed: % C 0.3; % H 0.63.This result is consistent with glycidyl modification, as shown informula (XVI), above.

2. Histidine Linkage: 0.5 g of D,L-histidine was dissolved in a mixtureof 4 ml of tetrahydrofuran and 6 ml of water. 1.2 g of glycidyl-modifiedsilica magnetic particles was added to the mixture; and the resultingsuspension was refluxed for 5 hr. After cooling to room temperature thereaction mixture was filtered, the solid washed once with 50 ml ofmethanol and 50 ml. of ethyl ether. The product was dried under vacuumin a desiccator over phosphorous pentoxide. Elemental analysis revealed:% C 0.44; % H 0.64; % N 0.0. This result is consistent with glycidyllinkage of histidine to the non-porous silica magnetic particles,according to formula (XVII), above.

B. Synthesis of Glycidyl-Histidine Modified Glass-Fibers

1. Glycidine Modification: 0.7 g of glass fiber filters (Ahlstrom-122;Ahlstrom Filtration, Inc., Helsinki, Finland.) were suspended in 15 mlof toluene, and 1.0 ml of 3-glycidylpropyltrimethoxysilane was added tothe suspension. The resulting mixture was incubated at room temperaturefor 48 hr. The solution was removed from the resulting modified glassfiber filter products by pipetting. The filter products were washedtwice with 30 ml of methylene chloride, then soaked in methylenechloride for 30 min, and washed two more times with 30 ml. each ofmethylene chloride. This process of soaking and washing was repeated.The filters were dried under vacuum on a roto-evaporator.

2. Histidine Linkage: 0.6 g of D,L-histidine was dissolved in a mixtureof 10 ml of tetrahydrofuran and 15 ml of water. This solution was addedto the filters and the resulting suspension was refluxed for 20 hr.After cooling to room temperature the liquids were removed from thereaction by pipetting and the filters were washed extensively with waterand with methanol. The washed filters were air dried overnight.Elemental analysis of the end product showed: % C 0.55; % H 0.16; % N0.0. These results are consistent with glycidyl-histidine linkage,according to formula (IV), above.

C. Synthesis of Glycidyl-Histidine Modified Silica Gel

1. Glycidine Modification: 10.0 g of Silica Gel 110 HP [ChromatographicSilica Grade 110 HP from W. R. Grace (Baltimore, Md.)] was suspended in45 ml of toluene, and 5.0 ml of 3-glycidylpropyl-trimethoxysilane wasadded to the suspension. The resulting mixture was placed on aroto-evaporator overnight. The reaction mixture was filtered and thesolid product was washed once with 20 ml of methylene chloride and oncewith 20 ml of ethyl ether. The product was dried under vacuum in adesiccator over phosphorous pentoxide. Elemental analysis: % C 7.75; % H1.67. These results are consistent with glycidine modification.

2. Histidine Linkage: 10 g of all of the above modified silica wassuspended in 30 ml of tetrahydrofuran and 50 ml of water. To thissolution 3.8 g of D,L Histidine was added and the resulting suspensionwas refluxed overnight (about 18 hr). After cooling to room temperaturethe reaction mixture was filtered, washed once with 200 ml of methanoland once with 50 ml of ethyl ether. The resulting product was driedunder vacuum in a desiccator over phosphorous pentoxide. Elementalanalysis revealed: % C 9.88; % H 1.92; % N 1.68. These results areconsistent with glycidyl-histidine modification, according to formula(IV), above.

Example 5 Preparation of Porous Silica Magnetic Urea-Linked pH DependentIon Exchange Particles A. Silica Magnetic Particles Linked to HistidineThrough Urea

1. Modification with Urea: 5 g of histidine ethyl ester dihydrochloridewas suspended in 50 ml of chloroform and 4.0 ml of triethylamine. 4.8 gof 3-isocyanatopropyltrimethoxysilane was added to this solutiondrop-wise, via an addition funnel, and the resulting silane/chloroformsolution was stirred overnight. 2.0 g of porous silica magneticparticles were suspended in 25.0 ml of the silane/chloroform solution,and this mixture was placed on a roto-evaporator for 20 hr. Theresulting reaction mixture was filtered, and the retentate, whichincluded silica magnetic particles modified in the reaction, was washedonce with 50 ml of chloroform and once with 50 ml of ethyl ether. Thewashed product was dried in a desiccator under vacuum over phosphorouspentoxide. Elemental analysis revealed: % C 2.38; % H 0.96; % N 0.81.These results are consistent with results one would expect from a silicamagnetic particles modified with urea.

2. 1.0 g of the modified particles was suspended in 5% HCl and stirredfor 4 hr. The particles were separated from the HCl solution, washedwith water, resuspended in 25 ml of water, and filtered. The retentate,which included the modified silica magnetic particles, was washed oncewith 50 ml of water, once with 50 ml of methanol, and once with 50 ml ofethyl ether. The washed solid was dried under vacuum in a desiccatorover phosphorous pentoxide. Elemental analysis showed: % C 1.59; % H0.84; % N 0.55. These results are consistent with what one would expectfrom a silica magnetic particle linked to histidine via urea, asillustrated in formula (XX), below:

wherein, R is —OH, —OCH₃, or —OCH₂CH₃.

B. Synthesis of Silica Magnetic Particles Linked to Histamine andPropionate

1. Synthesis of N-2-(4-Imidazole)-ethyl-N′-3-propyltriethoxysilylurea:4.5 g of histamine was suspended in 50 ml of Chloroform. 9.8 g. of3-Isocyanatopropyltrimethoxysilane was added drop-wise to thesuspension, via an addition funnel, and the resulting reaction stirredovernight. After this period the reaction was evaporated to dryness. Theproduct was not further purified. Results of analysis of thisintermediate product using nuclear magnetic resonance spectroscopy (NMR)were consistent with what one would expect fromN-2-(4-Imidazole)-ethyl-N′-3-propyltriethoxysilylurea. Specifically, NMR(CD3OD) results found were: 7.6 ppm (s, 1H); 6.8 (s, 1H); 4.7 (broad s,4H); 3.8 (q, 4H); 3.6 (q, 1H) 3.36 (t, 2H); 3.30 (m, 1H); 3.07 (t, 2H);2.72 (t, 2H); 1.55 (m, 2H); 1.2 (m, 6H).

2. Linkage of Histamine via Urea: 1.0 g of silica magnetic particles wassuspended in 10 ml of chloroform, and 1.2 g of theN-2-(4-Imidazole)-ethyl-N′-3-propyltriethoxysilylurea produced in step1, above, was added to the suspension. The resulting mixture was placedon a roto-evaporator for 48 hr. The reaction was filtered andresuspended in 40 ml of Chloroform. The solid was filtered and washedwith chloroform and ethanol. The solid was dried in a desiccator undervacuum over phosphorous pentoxide for 2 hr. Elemental analysis results(% C 5.46; % H 1.16; % N 2.35) were consistent the results one wouldexpect to obtain from silica magnetic particles modified with histamine.

3. Methyl Propionate Modification: 1 g of the entire amount of histaminemodified silica magnetic particles from step 2, above, was suspended in10 ml of toluene and 1.0 ml of 2-(carbomethoxy)ethyltrichlorosilane wasadded drop-wise with stirring. The resulting reaction mixture stirredfor 2 hr. After this time the solid was filtered and washed withchloroform and ethanol. The product was dried under vacuum for 1 hr in adesiccator over phosphorous pentoxide. Elemental analysis results (% C7.24; % H 1.52; % N 2.07) were consistent with methyl propionatemodification of histamine modified particles.

4. Removal of Methyl Group from the Propionate Residues: 1 g of silicamagnetic particles modified in Step 3 was suspended in 5% HCl andstirred for 4 hrs. The reaction products were separated from thesolution by filtration. The retentate of reaction product, whichincluded the modified particles, was washed with water and methanol. Thewashed product was dried under vacuum in a desiccator over phosphorouspentoxide. Elemental analysis results (% C 6.14; % H 1.37; % N 1.47)were consistent with silica magnetic particles linked to histaminethrough urea and also modified by propionate, according the formula(XXI), below:

wherein, R¹ and R³ are, independently, —OH, —OCH₃, or —OCH₂CH₃; R² is—(OSiR² ₂)_(y)—R², wherein y is at least 0; and R⁴ is —(OsiR³ ₂)_(z)—R³,wherein z is at least 0.

C. Synthesis of Silica Magnetic Particles Linked to Histidine andPropionate

1. Histidine was covalently attached to silica magnetic particles via aurea linkage, using a procedure similar to that used to attach histaminein part A of this Example, above.

2. The same final two steps used to covalently attach propionate to theurea-linked histamine particles in part B of the Example, above wereused to covalently attach propionate to the silica magnetic particleslinked to histidine via propionate.

Example 6 Preparation of Cleared Lysate of Plasmid DNA

E. coli bacteria cells, DH5α strain, were transformed with pGL3-ControlVector (Promega) plasmid DNA, and grown in an overnight culture of LuriaBroth (“LB”) medium at 37° C., then harvested by centrifugation.

The following solutions were used to prepare a lysate of the harvestedcells, as described below:

Cell Resuspension Solution

50 mM Tris-HCl, pH 7.5

10 mM EDTA

100 μg/ml DNase-free ribonuclease A (RNase A)

Wizard® Neutralization Buffer (Promega Corp.)

1.32M KOAc (potassium acetate), pH 4.8

Cell Lysis Solution

0.2M NaOH

1% SDS (sodium dodecyl sulfate)

A cleared lysate of the transformed cells was produced as follows:

1. The cells from 1 to 10 ml of bacteria culture were harvested bycentrifuging the culture for 1-2 minutes at top speed in amicrocentrifuge. The harvested cells were resuspended in 250 μl of CellResuspension Solution, and transferred to a microcentrifuge tube. Theresulting solution of resuspended cells was cloudy.

2. 250 μl of Cell Lysis Solution was then added to the solution ofresuspended cells and mixed by inversion until the solution becamerelatively clear, indicating the resuspended cells had lysed.

3. 350 μl of Wizard® Neutralization Buffer was added to the lysatesolution, and mixed by inversion. The lysate became cloudy after theNeutralization Solution was added.

4. The solution was then spun in a microcentrifuge at top speed (about12,000 G) for 10 minutes to clear the lysate.

Example 7 Isolation of Plasmid DNA using Porous Silica MagneticGlycidyl-Histidine pH Dependent Ion Exchange Particles

All preps were processed in 1.5 ml tubes, and all steps were performedat room temperature:

1. The cleared lysate from step 5 of Example 6 was transferred to aclean 1.5 ml tube containing 150 μl of an pH dependent porous silicamagnetic ion exchange particles (15 mg of particles) linked to histidinethrough a glycidyl moiety, wherein the particles prepared as describedin Example 3B. The resulting mixture of particles and solution wasvortexed, and incubated at room temperature for 5 minutes.

2. The silica magnetic ion exchange particles contained in the tube wereheld against the inner side-wall of the tube by magnetic force, whilethe tube cap and side-wall were washed with the lysate solution fourtimes by inversion, and allowed to sit for 1 minute at room temperature.The solution was removed and discarded.

3. The particles tube and cap were washed with 1.0 ml nanopure water.

4. Magnetic force was used to hold the silica magnetic particles in thetube while liquid in the tube was removed therefrom and from the tubecap. The liquid was discarded.

5. The particles were resuspended by vortexing in 300 ul of 66 mMpotassium acetate and 800 mM NaCl (pH 4.8). Step 3 was repeated.

6. Step 5 was repeated three times, for a total of four salt washes.

7. The silica magnetic particles remaining in the tube were resuspendedin 1.0 ml of nanopure water.

8. The silica magnetic ion exchange particles were separated from thewater by magnetic force. The tube cap and side-wall was washed withwater by tube inversion (4×), and allowed to sit 1 minute.

9. Liquid was removed from the tube and cap.

10. Steps 7-9 were repeated for a total of 2 washes, with water.

11. 100 ul of 10 mM Tris pH 8.0 was added to the tube to elute the DNA,and the tube was vortexed thoroughly.

12. The silica magnetic ion exchange particles were separated from theeluent by magnetic force, and the eluent removed to a clean tube.

Analytical analysis of the eluent from step 12 showed that plasmid DNAwas obtained which was relatively free of contaminating proteins orother nucleic acids. Specifically, analysis of the eluent using gelelectrophoresis according to the procedure set forth in Example 1,above, showed no RNA or chromosomal RNA contamination. Analysis of theeluent using absorption spectroscopy as described in Example 2, showedthe yield of pGL-3 plasmid DNA to be 30 μg. Absorbance ratio results(A₂₆₀/A₂₈₀ ratio of 1.84) indicated the plasmid DNA isolated accordingto the procedure described above was free of protein contamination.

Example 8 Isolation of Plasmid DNA from a Cleared Lysate usingGlycidyl-Histidine Glass Fibers

A cleared lysate from 5 ml of an overnight culture of DH5α cellstransformed with pGL3 Control Vector plasmid DNA was prepared asdescribed in Example 3. The cleared lysate was added to a columncontaining 42 mg of Ahlstrom 121 glass fiber modified byglycidyl-histidine, as described in Example 4B, above. After 10 minutesof binding time, the column was centrifuged to remove the alkalinelysate solution. The column was then washed using 700 μl of nanopurewater, which was removed by column centrifugation. This water wash wasrepeated twice (for a total of three washes). The DNA was eluted with100 μl of 10 mM Tris pH 8.0, and the solution collected into a 1.5 mltube by column centrifugation. The eluted DNA was examined by gelelectrophoresis according to the procedure set forth in Example 1, andno RNA or chromosomal DNA contamination was detected. Analysis by atomicabsorbsion spectroscopy showed a DNA yield of 36 μg, and an A₂₆₀/A₂₈₀ratio of 1.86.

The column was washed with 400 μl of 10 mM Tris pH 8.0 (which wasremoved by column centrifugation), and washed again with 2×700 μl of 100mM Tris, 2.0M NaCl (also removed by column centrifugation). The columnwas then washed with 700 μl of nanopure water, (removed by columncentrifugation), and air dried for 12 hours at room temperature.

The column was reused, following the same procedure as outlined above.The resulting DNA again showed no visible RNA by gel electrophoresis,and a DNA yield of 30 ug and an A₂₆₀/A₂₈₀ ratio of 1.84.

Example 9 Isolation of Plasmid DNA from a Cleared Lysate usingNon-Porous Glycidyl-Histidine Ion Exchange Particles Functionalized withGlycidyl Histidine

A cleared lysate of DH5α cells transformed with pGL3 Control Vectorplasmid DNA was prepared as described in Example 6, except 500 ul ofWizard® Neutralization Buffer was added to the lysed cells in step 3,rather than 350 ul. Plasmid DNA was isolated from the cleared lysateusing non-porous glycidyl-histidine silica particles prepared asdescribed in Example 4A, as follows:

The cleared lysate was combined with 15 mg of the glycidyl-histidinenon-porous silica particles in a 3 ml syringe barrel, and allowed to sitat room temperature for 1 hour.

The lysate was then pushed through the syringe barrel, by positivepressure. Two 1.0 ml washes with nanopure water were performed, usingpositive pressure to remove the liquid. Then 100 ul of 10 mM Tris, pH8.0 was used to elute the DNA. The eluted DNA was removed by positivepressure into a clean 1.5 ml tube.

Analysis by gel electrophoresis, according to the procedure of Example1, showed the eluent to contain supercoiled plasmid DNA, with noevidence of contamination with chromosomal DNA or RNA. Absorptionanalysis of the eluent, according to the procedure of Example 2, showeda yield of 10 mg of DNA, and an absorbance ratio of A₂₆₀/A₂₈₀ of 1.61.

Example 10 Isolation of Plasmid DNA from a Cleared Lysate using PorousSilica Magnetic Glycidyl-Alanine

Plasmid DNA was isolated from DH5α E. coli bacteria cells transformedwith pGEM-3Zf+ DNA, as follows. Preps were processed in 1.5 ml tubes.All steps were performed at room temperature, except where indicatedotherwise below.

1. 2.5 ml of Wizard® Resuspension Solution was added to a 50 ml pelletof transformants, and vortexed vigorously to resuspend cells.

2. 265 μl of resuspended cells were added to two tubes.

3. 250 μl of Wizard® Lysis Buffer was added per tube, and gently mixedto avoid sheering genomic DNA.

4. 350 μl of Wizard® Neutralization Solution was added per tube, andmixed gently.

5. The tubes were centrifuged at 14 k rpm for 10 minutes.

6. The cleared solution was removed and placed in a clean 1.5 ml tubecontaining 150 ul of 100 mg/ml (15 mg) silica magnetic glycidyl-alanineparticles prepared as described in Example 3C, above. The resultingmixture was vortexed, and incubated 5 minutes.

7. The particles were separated from the mixture, using a magneticseparator. The tube caps were washed by tube inversion (4×), andincubated 1 minute.

8. Liquid was removed from tubes, including caps.

9. Tubes were washed with 1.0 ml of nanopure water.

10. Steps 7 and 8 were repeated.

11. Steps 9 and 10 were repeated twice, for a total of 3 washes.

12. An elution buffer of 100 μl of 20 mM Tris-HCl, pH 9.5, was added toeach tube. The particles and buffer were mixed well to allow plasmid DNAwhich had adsorbed to the particles to elute therefrom.

13. The particles were separated from the resulting eluent by magneticforce. The eluent solution in each tube was transferred to a clean tube.

Duplicate isolations conducted according to the procedure describedabove yielded 21.7 μg (A260/280 of 1.86) and 16.1 μg (A260/280 of 1.89)of plasmid DNA. No RNA was visible by analysis using gelelectrophoresis.

Example 11 Comparison of Counterion Conditions Required to Elute PlasmidDNA from Silica Magnetic Urea-Linked Histamine, and Silica MagneticUrea-Linked Histamine and Propionate Bimodal Ion Exchange Particles atVarious pH's

The minimum amount of sodium chloride and a buffer required to eluteplasmid DNA from each of two different types of silica magnetic pHdependent ion exchange particles was assayed at each of severaldifferent pH's, according to the following procedure. One of the twotypes of particles used in this assay was silica magnetic particleslinked to histidine through a urea residue (referred to in the presentExample as “urea-histidine IE particles”), prepared as described inExample 5A, above. The other type of particle used in this Example wassilica magnetic particles linked directly to propionate and linked tohistamine through a urea residue (hereinafter,“bimodal-histamine-propionate IE particles”) prepared as described inExample 5B, above. Elemental analysis of thebimodal-histamine-propionate IE particles showed 260 μmoles of histamineand 900 μmoles of propionate.

Cleared lysates were prepared from the DH5α strain of E. coli bacteriacells transformed with pGL3-Control Vector (Promega), as described inExample 6, above, modified as follows. Cells from 50 ml of an overnightculture of the transformants were harvested by centrifugation, andresuspended in 2.5 ml of Wizard® Resuspension Solution. The cells werelysed by adding 2.5 ml of Wizard®Lysis Solution to the resuspendedcells. 3.5 ml of Wizard® Neutralization Solution was added to theresulting lysate. The lysate was cleared by centrifugation, and thesupernatant transferred to a sterile 50 ml tube.

The urea-histidine IE particles and bimodal-histamine-propionate IEparticles were tested and compared to one another for their capacity tobind to and release plasmid DNA from the cleared lysate prepared asdescribed immediately above. The elution solution used to isolateplasmid DNA with each of the two types of particles varied, with a pHranging between pH 4.2 and 9.5:

1. 700 μl of the cleared lysate was added to each 1.5 ml microfuge tubein each of four sets of two samples for each of the two types ofparticles tested. Each 1.5 ml microfuge tube contained 150 μl of eitherof the two types of particles (15 mg). Each tube was capped and mixed byinversion. The resulting suspension was incubated at room temperaturefor 5 minutes.

2. The particles and solution were separated by magnetic force, and thesolution removed from each tube. 1.0 ml of nanopure water was added toeach tube, used to wash the particles, separated from the particles bymagnetic force, and removed from the tube. For all the sets of samplesexcept those to be eluted at a pH of below pH 5 (e.g. samples to beeluted at 4.2 or 4.8), the water wash was repeated.

3. The particles were resuspended in 300 μl of the putative elutionsolution. The particles were magnetically separated, and the solutioncarefully removed to a clean 1.5 ml tube. The salt concentration of theelution solution has modified, by addition of either water or 5M NaCl,to a final concentration of 1 M NaCl. The DNA (if present) wasconcentrated by precipitation with 1.0 ml of −20° C. ethanol. The DNAwas pelleted by centrifugation in a microfuge at 12,000×g for 10minutes. The pellets were dried to remove ethanol, and resuspended in100 μl of 10 mM Tris HCl pH 9.5.

4. The particles remaining from step 3 were washed once with 1.0 mlnanopure water, and then treated as the particles at the beginning ofstep 3. In this way, a variety of elution solutions were tested, in astepwise fashion, using the same DNA bound particles.

5. For elution conditions above pH 8.0, 100 μl of 10 mM Tris HCl wasused in the case of the bifunctional IE particles. Similar testing ofthe urea-histamine IE particles showed no DNA elution at 10 mM Tris HCl,even at pH 9.5. The eluted DNA was examined by gel electrophoresis todetermine the minimum counterion concentration need for DNA elution.Once the approximate concentration was determined, the procedure wasrepeated to confirm the concentration of potassium acetate and NaCl atpH 4.8, and the concentration of Tris HCl and NaCl at pH 7.3, and pHsabove 7.3.

Elution conditions used on each set of samples prepared as describedabove are shown in Table 1, below:

TABLE 1 pH Urea-Histidine IE Particles Bifunctional IE Particles 4.2  33mM KOAc/2.15M NaCl 4.8  33 mM KOAc/1.7M NaCl 7.3 100 mM Tris HCl/600 mMNaCl 100 mM Tris/300 mM NaCl 8.0 100 mM Tris/300 mM NaCl 100 mM Tris/noNaCl 8.7 100 ul of 10 mM Tris HCl 9.5 100 ul of 50 mM Tris HCl

The results above demonstrate that the addition of propionate groups tourea-histidine IE particles reduces the amount of counterionconcentration required to elute DNA from such particles.

Example 12 Isolation of PCR Amplified DNA from UnincorporatedNucleotides and Primers, Using Non-Porous Silica MagneticGlycidyl-Histidine pH Dependent Ion Exchange Particles. SimilarPurification of PCR Amplified DNA Using Porous Silica Magnetic GlycidylCysteine pH Dependent Ion Exchange Particles

The human APC (Adenomatous Polypoptosis Coli) gene was amplified in aPCR amplification reaction, wherein human genomic template DNA was addedto a reaction mix containing:

40 ul 10×AmpliTaq® PCR buffer (no Mg++) [Perkin Elmer];

40 ul 25 mM MgCl₂;

13 ul 10 mM dNTP mix;

13 ul APC primers (50 μmoles/μl), with nucleotide sequences: 5′GGA TCCTAA TAC GAC TCA CTA TAG GAA CAG ACC ACC ATG CAA ATC CTA AGA GAG AAC AACTGT C3′ SEQ ID NO:1, and 5′CAC AAT AAG TCT GTA TTG TTT CTT 3′; SEQ IDNO:2

6.4 ul AmpliTaq® [Perkin Elmer]; and

273.6 ul of nanopure water [total=392 μl].

The amplification reaction was run for 35 cycles on a Perkin Elmer 4800thermocycler. A 1.8 kb DNA product was the result of the amplification.

The resulting PCR amplified gene was isolated from other components inthe reaction mix, above according to the following isolation procedure:

1. 20 μl of the PCR reaction mix was added to 200 μl of 66 mM KOAc+900mM NaCl, pH 4.8, and mixed. Then, 20 μl (2 mg) of non-porousglycidyl-histidine silica magnetic particles was added.

2. After mixing, the solution was incubated for 5 minutes at roomtemperature. The particles were separated by use of a magneticseparator, and the solution was removed to a clean 1.5 ml tube.

3. The particles were resuspended by vortexing in 200 μl of nanopurewater, and separated from the resulting solution. The particles wereseparated using a magnetic separator, the cap and side-wall of the tubewere washed by inverting the tube, and the solution was removed from thecap and tube, and placed in a clean 1.5 ml tube.

4. The PCR amplified DNA was eluted in 20 μl of 10 mM Tris HCl pH 8.0.The particles were separated by magnetic force and the eluted DNA wasremoved to a clean 1.5 ml tube.

5. Using gel electrophoresis (see Example 1), the solutions obtainedfrom steps 2, 3, and 4 were compared with a sample of the original PCRreaction. The solution from steps 2 showed no visible PCR amplified DNA.The solution from step 2 showed a small amount (about 10% of the initialamount) of the PCR DNA. The solution from step 4 showed an amount of PCRDNA>80% of the initial amount in the reaction mix, and no visibleunincorporated primers and nucleotides, as seen in the initial PCRreaction solution.

The same procedure was followed using MagneSil™ (no histidine ligand)porous particles, and resulted in no visible DNA at the end of step 4.

The same amplification mixture was purified using porous silica magneticglycidyl-cysteine pH dependent ion exchange particles and using silicamagnetic particles (as a control), according to the following procedure:

1. Three 1.5 ml tubes were set up with 20 ul of amplification mixturemixed with 200 ul of 33 mM KOAc/400 mM NaCl, pH 4.8. To tubes 1 and 2,20 μl (2mg) of Mag-IE-glycidyl-cysteine was added and mixed. To tube 3,20 μl of Magnesil™ particles was added and mixed.

2. Each tube was incubated 10 minutes at 20° C., and the particles ineach tube separated from the solution in each tube by magnetic force,for 2 minutes.

3. The solution from each tube was removed. The sololutions from tubes 1and 2 were processed according to steps 4-5, below. The particles intube 3 were resuspended in 33 mM KOAc/400 mM NaCl, pH 4.8, magneticallyseparated for 2 minutes, and the solution removed and processedaccording to steps 4-5, below.

4. The particles were resuspended in 200 ul of nanopure water,magnetically separated, and the solution removed from the tube.

5. DNA was eluted in 20 ul of 50 mM Tris HCl pH 9.5

Aliquots of the original amplification reaction products and of theeluents from Magnesil™ (tube 1, above) and fromMag-IE-glycidyl-histidine (tubes 2-3 above) were analyzed by gelelectrophoresis, as described in Example 1, above. The resulting gel wasstained with ethidum bromide, and a photograph thereof taken under UVlight. FIG. 1 shows the gel, with:,

Lane 1: Eluent from the Magnesil™ particles (tube 1, above).

Lane 2: Eluent from the Mag-IE-glycidyl-histidine particles (tube 2,above), with no wash step prior to transfer of the particles from theamplification reaction solution to nanopure water in step 4, above.

Lane 3: Eluent from the Mag-IE-glycidyl-histidine particles (tube 3,above), after washing the particles in 33 mM KOAc/400 mM NaCl, pH 4.8prior to transfer to nanopure water in step 4, above.

Lane 4: Aliquot of the amplified DNA reaction mixture.

Note that the amplified DNA reaction mixture includes bands other thanthe desired amplification product. The Magnesil™ particles appear tohave failed to isolate any detectable quantity of the amplified DNAfragments, as no bands are visible in lane 1 of FIG. 1. Both isolationprocedures with Mag-IE-glycidyl-histidine produced amplified DNAisolated from low molecular weight species (the band below the primaryband in lane 4). However, considerably more amplified DNA was producedfrom tube 2, without the additional wash step, than was isolated fromtube 3 with the additional wash step.

Example 13 Isolation of Human Genomic DNA from Buccal Swabs, UsingNon-Porous Silica Magnetic Glycidyl-Histidine Particles

Genomic DNA was isolated from buccal swabs using non-porous silicamagnetic glycidyl-histidine ion exchange particles, synthesized asdescribed in Example 3B, above, as follows:

Tissue samples were obtained from two inner cheek areas of humansubjects, using cotton swabs (buccal collection), and the swabs wereallowed to sit at room temperature for 10 minutes, with occasionalswirling, in 700 μl of a cell lysis buffer (75 mM Na Citrate pH 5.0/1.5%Tween) in a 1.5 ml microfuge tube. The swabs were removed and the liquidin the swabs was pressed out by running it over the opening of the tube,pressing the swab into the interior side of the tube.

30 μl of proteinase K (18 mg/ml) was added to each tube, and 50 μl (5mg) of non-porous silica magnetic glycidyl-histidine particles was addedper tube, and mixed well. Samples were incubated at room temperature for5 minutes, with occasional mixing by tube inversion.

The tubes were placed on a magnetic rack to allow separation of thesolution and particles, and the solution was removed from the tube.

The particles were washed twice with 1.0 ml of nanopure water. Afterremoval of the second 1 ml of water, the DNA was eluted in 40 μl of 20mM Tris HCl pH 9.5, at 65° C. for 5 minutes.

Magnetic force was used to separate the particles from the eluted DNA.

The eluted DNA was examined by gel electrophoresis, as described inExample 1, above, and compared to a control sample of a known amount ofgenomic DNA to estimate the quantity of DNA eluted. Each 40 μl sample ofeluted DNA was found to contain greater than 100 ng of genomic DNA.

Example 14 Comparison of Counterion Conditions Required to Elute PlasmidDNA from Silica Magnetic Urea-Histidine pH Dependent Ion ExchangeParticles and Silica Magnetic Urea-Histidine Propionate Bimodal pHDependent Ion Exchange Particles

The minimum amount of sodium chloride and a buffer required to eluteplasmid DNA from each of two different types of silica magnetic pHdependent ion exchange particles was determined at each of several pH's,according to the following procedure. Silica magnetic urea-histidine IEparticles prepared as described in Example 5A, and silica magneticbimodal urea-histidine-propionate IE particles prepared as described inExample 5C were used to isolate plasmid DNA from a cleared lysate, asfollows.

Cleared lysates were prepared as described in example 11. The procedurefor comparing the elution profiles of the two particles was as describedin example 11. The pHs tested were 4.8, 7.3, and 9.5. The resultsobtained are shown in Table 3, below:

TABLE 3 MAGNETIC PARTICLE ELUTION/NON-ELUTION AND pH CONDITIONSCONDITIONS Urea-histidine IE particles, DNA eluted in 33 mM KOAc/1.45MNaCl, pH 4.8 did not elute in 33 mM KOAc/1.40M NaCl Bimodalurea-histidine- DNA eluted in 33 mM KOAc/0.80M NaCl, propionate IEparticles, did not elute in 33 mM KOAc/0.70M NaCl pH 4.8 Urea-histidineIE particles, DNA eluted in 100 mM Tris HCl, pH 7.3 did not elute in 80mM Tris HCl Bimodal Urea-histidine- DNA eluted in 60 mM Tris HCl,propionate IE particles, did not elute in 50 mM Tris HCl pH 7.3Urea-histidine IE particles, Did not elute in 100 ul of 10 mM Tris HCl,pH 9.5 but eluted in 100 ul of 100 mM Tris HCl Bimodal Urea-histidine-Eluted in 100 ul of 10 mM Tris HCl propionate IE particles, pH 9.5

By spectrophotometric analysis, the elutions in 100 ul of 10 mM Tris HClat pH 9.5 yielded 30 μg (A₂₆₀/A₂₈₀ of 1.78) of DNA for the bimodalurea-histidine-propionate IE particles and less than 2 μg of DNA for theurea-histidine IE particles. No DNA was detected on analysis of theeluent from the urea-histidine IE particles, by gel electrophoresis.

The results above indicate that the addition of propionate to theurea-histidine particles lowered the needed concentration of counter-ion(chloride) required for elution of the DNA at pH 4.8, 7.3 and 9.5.

2 1 64 DNA Homo sapiens Oligonucleotide primer of the AdenomatousPolypoptosis Coli gene 1 ggatcctaat acgactcact ataggaacag accaccatgcaaatcctaag agagaacaac 60 tgtc 64 2 24 DNA Homo sapiens Oligonucleotideprimer of the Adenomatous Polypoptosis Coli gene 2 cacaataagt ctgtattgtttctt 24

What is claimed is:
 1. A pH dependent ion exchange matrix, comprising: asolid support, and a plurality of ion exchange ligands, each first ionexchange ligand comprising: a cap comprising an amine with a pK of lessthan about 9; a spacer covalently attached to the cap, the spacercomprising a spacer alkyl chain with an amine terminus and an acidicmoiety covalently attached to the spacer alkyl chain, wherein the acidicmoiety is a carboxyl residue; and a linker comprising a linker alkylchain covalently attached to the solid support at a first end of thelinker alkyl chain and covalently attached to the amine terminus of thespacer at a second end of the linker alkyl chain; wherein the matrix hasa capacity to adsorb to a target nucleic acid at a first pH, and torelease the target nucleic acid at a desorption pH which is higher thanthe first pH.
 2. The matrix of claim 1, wherein the solid support is asilica based material.
 3. The matrix of claim 2, wherein the silicabased material is a glass fiber.
 4. The matrix of claim 2, wherein thesilica based material is a silica gel particle.
 5. The matrix of claim4, wherein the silica gel particle is paramagnetic.
 6. The matrix ofclaim 4, wherein the silica gel particle is porous.
 7. The matrix ofclaim 4, wherein the silica gel particle is non-porous.
 8. The matrix ofclaim 1, wherein the cap further comprises an aromatic hydrocarbon ring.9. The matrix of claim 8, wherein at least one member of the aromatichydrocarbon ring is the amine with a pK of less than about
 9. 10. Thematrix of claim 9, wherein the aromatic hydrocarbon ring is selectedfrom the group consisting of pyridine, and imidazole.
 11. The matrix ofclaim 1, wherein the amine with a pK of less than about 9 has a pK of atleast about 4 and up to about
 6. 12. The matrix of claim 1, wherein thespacer alkyl chain comprises two (2) to five (5) carbon atoms.
 13. Thematrix of claim 1, wherein the spacer is selected from the groupconsisting of cysteine and alanine.
 14. The matrix of claim 1, whereinthe aromatic hydrocarbon covalently linked to the spacer define a basicamino acid moiety selected from the group consisting of histidine andhistamine.
 15. The matrix of claim 1, wherein the linker alkyl chaincomprises three (3) to eight (8) carbon atoms.
 16. The matrix of claim1, wherein the linker alkyl chain includes at least one member selectedfrom the group consisting of oxygen and amine.
 17. The matrix of claim1, wherein the linker is selected from the group consisting of: glucidyland urea.
 18. The matrix of claim 1, wherein the matrix is an anionexchanger capable of exchanging with the target nucleic acid at thefirst pH, and the matrix has a net neutral or negative charge at thedesorption pH.
 19. The matrix of claim 1, wherein the desorption pH isat least about 4.0 and up to about pH 10.0.
 20. The matrix of claim 1,wherein the matrix can be reused through at least two cycles ofadsorption of the target nucleic acid to the matrix at the first pH andof release from the matrix at the desorption pH.
 21. The matrix of claim1, wherein the plurality of ion exchange ligands covalently attached tothe solid support has a density of at least about 25 μmol per gram dryweight of the matrix and no greater than about 500 μmol per grain dryweight of the matrix.
 22. A pH dependent ion exchange matrix,comprising: a solid support, and a plurality of ion exchange ligands,each first ion exchange ligand comprising: a cap comprising an aminewith a pK of less than about 9; a spacer covalently attached to the cap,the spacer comprising a spacer alkyl chain with an amine terminus and anacidic moiety covalently attached to the spacer alkyl chain; and alinker comprising a linker alkyl chain covalently attached to the solidsupport at a first end of the linker alkyl chain and covalently attachedto the amine terminus of the spacer at a second end of the linker alkylchain; wherein the matrix has a capacity to adsorb to a target nucleicacid at a first pH, and to release the target nucleic acid at adesorption pH which is higher than the first pH, and wherein the capfurther comprises an aromatic hydrocarbon ring.
 23. The matrix of claim22, wherein at least one member of the aromatic hydrocarbon ring is theamine with a pK of less than about
 9. 24. The matrix of claim 22,wherein the aromatic hydrocarbon ring is selected from the groupconsisting of pyridine, and imidazole.
 25. A pH dependent ion exchangematrix, comprising: a solid support, and a plurality of ion exchangeligands, each first ion exchange ligand comprising: a cap comprising anamine with a pK of less than about 9; a spacer covalently attached tothe cap, the spacer comprising a spacer alkyl chain with an amineterminus and an acidic moiety covalently attached to the spacer alkylchain; and a linker comprising a linker alkyl chain covalently attachedto the solid support at a first end of the linker alkyl chain andcovalently attached to the amine terminus of the spacer at a second endof the linker alkyl chain; wherein the matrix has a capacity to adsorbto a target nucleic acid at a first pH, and to release the targetnucleic acid at a desorption pH which is higher than the first pH, andwherein the aromatic hydrocarbon covalently liked to the spacer definesa basic amino acid moiety selected from the group consisting ofhistidine and histamine.